This proposal will explore the possible involvement of RNA self-splicing in genetic regulatory networks of bacteriophage. 1. In vitro splicing assays will be carried out to determine if guanosine-like molecules, that are found in bacterial cells, can compete for the ribozyme active site and inhibit splicing. 2. Splicing assays will also be carried out in vivo, under a variety of conditions of cell growth, to determine if anaerobiosis, unbalanced growth, or physiological stress can result in modulation of the splicing reaction. 3. The organization of the phage introns suggests that there may be coupling between translation of the genes contained within the introns, and relative splicing efficiency. This model will be tested by in vitro mutagenesis of the introns (to produce various levels of translation), to test whether there is an inverse relationship between splicing and translation. 4. A combination of classical and molecular genetic methods will be applied to selection of intron mutations and second site revertants. This method will be used to determine the molecular interactions that determine splice site specificity.